Parallel and cross-resistances of clinical yeast isolates determined by susceptibility pattern analysis

For calculated initial antifungal therapy, knowledge on parallel and cross-resistances are vitally important particularly in the case of multiresistant isolates. Based on a strain collection of 1,062 yeast isolates from a German/Austrian multicentre study, susceptibility pattern analysis (SPA) was used to determine the proportion of parallel and cross-resistances to eight antifungal agents (AFAs) encompassing flucytosine, amphotericin B, azoles (fluconazole, voriconazole and posaconazole) and echinocandins (caspofungin, micafungin and anidulafungin). A total of 414 (39.0%) isolates were resistant for one or more of the AFAs. Resistance to one AFA was shown for 18.1% of all isolates. For 222 isolates (20.9%), resistance to two to seven AFAs was noted (7.7%; 7.7%; 3.6%; 1.0%; 0.7% and 0.2% to 2, 3, 4, 5, 6 and 7 antifungal compounds, respectively). Partial parallel resistances within the azole and echinocandin classes, respectively, were found for 81 (7.6%) and 70 (6.6%) isolates. Complete parallel resistances for azoles, echinocandins and combined for both classes were exhibited by 93 (8.8%), 18 (1.7%) and 6 (0.6%) isolates, respectively. Isolates displaying cross-resistances between azoles and echinocandins were infrequently found. Highly resistant isolates (resistance to ≥6 AFAs) were almost exclusively represented by Candida albicans. Highly standardized testing of AFAs in parallel and from the same inocula followed by SPA allows detailed insights in the prevalence and distribution of susceptibility patterns of microbial isolates.


Background
Treatment options for invasive fungal infections are restricted by a very limited number of applicable antifungal agent (AFA) classes. Due to extensive worldwide use of fluconazole in the past decades, azole resistance has been significantly emerged, often associated with clinical failure [1], [2], [3], [4]. Also for the other AFA classes, resistances with substantial consequences for treatment and patient outcome have been increasingly reported [5], [6], [7], [8], [9], [10]. There is mounting evidence that the serious phenomenon of multi-resistance has reached also infections due to fungal pathogens. While reports on testing susceptibilities of yeast isolates to individual antifungals are available for many parts of the world [11], [12], [13], [14], systematic data on parallel and cross-resistances of Candida and other yeast isolates towards azoles, echinocandins, polyenes and flucytosine are still rare [15]. Of note, the terms "parallel resistances" and "cross-resistances" are often undifferentiated and/or varyingly used today. Here, parallel resistance (PR) was defined as resistance of a given isolate to all antifungal agents within an antifungal class and cross-resistance (CR) as resistance of a given isolate to antifungal agents belonging to different classes of antifungals. Previously, we have analyzed the AFA susceptibilities of 1,062 yeast isolates recovered from clinical specimens within a collaborative study including 17 participating medical centres mainly by standard susceptibility testing analyses [12]. Here, susceptibility pattern (SP) analysis (SPA) was applied allowing a highly standardized analysis and true comparison of antifungal susceptibilities based on determined individual SP of each single isolate [16]. Based on this, we determined the proportion of parallel, cross-and multi-resistances for the clinically most prevalent Candida species, but also for rare yeast species

Yeast isolates
A total of 1,062 clinical yeast isolates (species distribution, see Table 1) were recovered from clinical relevant routine samples of hospitalized patients and tested for susceptibility within a German/Austrian collaborative study comprising 17 study centres [12]. Details regarding species-specific resistance profiles and AFA-related resistance prevalences have been previously reported [12].

AFA and susceptibility testing
Susceptibility testing was performed as previously published according to DIN (Deutsches Institut für Normung e.V., i.e. the German Institute for Standardization) [12].
Statistical analysis was performed with SAS ® software (SAS Institute, Cary, NC, USA). The antilog of the calculations was displayed as MICs from calculated results. Differences were assessed by using Chi squared test; P values lower than 0.05 were considered statistically significant.

MICs and MIC interpretive criteria
Details of the in vitro susceptibilities of yeast isolates collected within the multicenter study towards eight antifungal agents have been reported before [12].  [21], [22]. CLSI criteria were applied for FCY and VOR [23]. Species-specific breakpoints were not applied. SPs representing sequences of interpretative categories (S, I or R) in a prefixed order of the test results were determined by SPA as described, here in an adaptation for fungal microorganisms [16]. An SP as applied here contains the number of members of antifungal class and the S-I-R categorization (e.g. for a given isolate with complete azole resistance to FLC, POS and VOR: 3R=R-R-R or for an isolate tested susceptible to all eight AFAs included: 8S=S-S-S-S-S-S-S-S).

Resistance definitions
Definitions were applied according to DIN 58940-1 and as published elsewhere [12], [17]. Briefly, multiple resistance (MR) was defined in this study, when two or more antifungal agents independently of any substance class were tested resistant in the same isolate, i.e. representing a random susceptibility pattern. Parallel resistance (PR) was defined as resistance of a given isolate to all (complete PR) or more than one, but not all (partial PR) AFAs within a class of antifungals. Cross-resistance (CR) was defined as resistance of a given isolate to two or more AFAs belonging to different classes of antifungals.

Results
A total of 1,062 clinical yeast isolates (C. albicans, n=573; 54.0%; non-albicans Candida spp., n=473; 44.5%; other yeasts, n=16; 1.5%; further details in Table 1) were enrolled for SPA evaluating their parallel and cross-resistance patterns. Unless otherwise stated, all results given in the text and the tables are based on the application of YST medium and endpoint reading after 48 h. For FCY, FLC, VOR and POS, a threefold categorization (S, I and R) was used and a twofold categorization (S and R) was applied for AMB, CAS, MCA and ANI. Thus, the individual SP of a given isolate represents one SP out of a variety of 1,296 theoretically possible SPs. However, only a limited amount of SPs were found, nevertheless, demonstrating numerous parallel and/or cross-resistant strains. Consequently, only a selection of relevant data restricted (i) to clinically most prevalent Candida species or (ii) to rare yeast species with noticeable SPs has been included in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6 and Table 7 Table 2). Considering all AFAs tested, only five (0.5%) isolates were characterized by exclusive resistance to VOR (Table 2).    (Table 4). SPA results for rare yeast species, defined in this study as Candida and non-Candida yeast species comprising equal or less than five isolates, are given in Table 5. AMBresistant isolates (each one isolate) were found for Candida lipolytica, C. melibiosica, C. sake and Geotrichum candidum. One C. melibiosica isolate showed complete parallel resistance to all azoles, but was susceptible to AMB, FCY and all echinocandins. The two Candida norvegensis isolates were tested resistant and intermediate, respectively, to FLC, but showed each a one-step more susceptible categorization towards VOR and POS. Both C. sake isolates offered resistances to echinocandins, one with complete parallel resistance and one remaining susceptible to MCA. A full echinocandin resistance was also noted for the G. candidum and Geotrichum capitatum isolates. The Kodamaea ohmeri isolate was tested susceptible to all AFAs with the exception of FLC (tested intermediate). Overall, complete susceptible SPs to all AFAs tested (8S-SP) were shown for 519/1062 isolates (48.9%) whereas 414 (39.0%) isolates were characterized by resistance to at least one of the AFAs included (≥1R) (Figure 1). The remaining 129 isolates (12.1%) without resistances demonstrated in one or more cases intermediate susceptibilities distributed to seven different SPs.
Comparing the proportion of a species within the total amount of study isolates versus its proportion within those isolates exhibiting a complete susceptible phenotype (8S SP), C. albicans isolates showed significantly higher  7R) were noted. The distribution of isolates exhibiting resistance to ≥2 AFAs was as follows: 2R, n=82 (7.7%); 3R, n=82 (7.7%); 4R, n=38 (3.6%); 5R, n=11 (1.0%); 6R, n=7 (0.7%), and 7R, n=2 (0.2%). SPs with ≥5R reflecting pronounced multi-resistance are given in Table 6 and Figure 1. Of 140 isolates (13.2%) characterized by a 3-7-fold AFA resistance, 97 (9.1%) possessed a complete parallel resistance consisting of 79 (7.4%), 12 (1.1%) and 6 (0.6) isolates showing this SP against all azoles, echinocandins and both AFA classes, respectively. Of note, highly resistant isolates exhibiting 7R-and 6R-patterns were almost exclusively represented by C. albicans with one exception by C. guilliermondii. The two 7R C. albicans strains were still susceptible to flucytosine ( Table 6). The proportion of still susceptible AFAs in relation to AFAstratified resistances has been calculated in Table 7 for the clinically most relevant Candida species. As shown in Table7 , FLC-resistant C. albicans isolates (n=46) displayed susceptibility to one of the echinocandins in more than 76% (CAS, 76.1%; ANI, 78.3% and MCA, 84.8%), but only 13.0% of these FLC-resistant isolates were also susceptible to VOR. Noteworthy, for C. glabrata, this analysis demonstrated that almost all FLC-(n=55), VOR-(n=35) and POS-(n=104) resistant isolates were tested susceptible to all echinocandins with the exception of two CAS-resistant isolates. While those C. krusei isolates tested resistant to FCY, AMB and/or one of the azoles revealed susceptibility to all echinocandins, respective C. parapsilosis isolates varied in the echinocandin susceptibility (Table 7). Except one ANI-resistant C. tropicalis isolate, almost all isolates of this species exhibiting complete parallel resistance to the azole class were tested susceptible to all echinocandins included (Table 3 and Table 7).

Discussion
In addition to antibiotic resistance towards bacterial and other pathogens, nowadays, also resistance to AFAs has emerged as one of the international health challenges to be addressed. AFA-resistant phenotypes may develop in yeast populations due to mutations, selection processes and alternative mechanisms (e.g. biofilm formation) and a priori-resistant species and strains exist [24]. Moreover, recombination may play also a role in fungi [25]. The fundamental "answer" of the fungal pathogens to the selection pressure by an increasing use of AFAs, however, is represented by shifts in the species and strain distribution towards those species characterized by intrinsic resistances or increased capabilities to express resistance mechanisms [25]. While a shift toward infections caused by non-albicans Candida species have been globally reported [26], [27], [28], a systematic review by Falagas et al. covering the period between 1996 and 2009, showed significant geographic, study design and setting variations of the relative frequency of Candida spp. among cases of candidemia in different parts of the world, consequently, local epidemiological data continue to be of major significance [29].
Here, eight AFAs were tested in parallel, at the same time, in same assay, with the same inocula, thus, all assayspecific parameters were equal for all AFAs allowing a unique, highly standardized evaluation of the isolates' susceptibilities. Considering pharmacological and pharmacodynamic aspects by a clinical breakpoint based categorisation (S-I-R), the results were arranged to individual SPs reflecting a resistance "fingerprint" for each single isolate, but embedded in the analysis of a large, recent multicentre isolate collection. Defining a fixed AFA sequence for SPA, SPs of different isolates can be easily compared and the frequencies of different SPs are determinable. Depending on the number of AFAs tested in parallel and the amount of parameters compared (e.g. methods, endpoint determinations, breakpoints, MICcategorizations), a multitude of different SPs may have gained allowing detailed analyses of susceptibility distributions, for example, dependent on the methodical approaches used. While standard descriptive methods and resulting data are the essential basis for questioning resistance preferentially for epidemiologically aspects, clinically and therapeutically relevant problems require comparative susceptibility evaluation methods. For this purpose, comparative AFA evaluation of individual isolate-specific susceptibilities may be useful, e.g. for determination of the prevalence of multi-resistant pathogens or to discover the susceptibility loss to complete AFA classes (e.g. azoles and echinocandins). For that purpose, SPA may act as useful tool allowing analyses of large strain collections down to the level of individual isolate-specific conclusions [30]. The data gained in this study by analyses of crosssusceptibility and -resistance patterns, respectively, are in particular relevant for treatment-related decisions. Here, of utmost clinical interest are those isolates exhibiting a complete parallel resistance to the entire azole class, in particular, if this is accompanied by a partial or, even worse, a complete echinocandin parallel resistance (Table 5 and Table 6). In contrast to earlier presumptions that no complete echinocandin cross-resistance exists or that there would be only a low potential for the resistance development to echinocandins [8], [31], we could clearly demonstrate by SPA approach that complete parallel resistance within all echinocandins occurs, here found in 1.7% of the clinical routine isolates included. In comparison, the amount of complete parallel resistance within the azole class was 8.8% characterized by speciesspecific variations. Although it is reported that clinical isolates with high echinocandin MICs tend to be low [32], isolates with echinocandin MICs of ≥4 mg/L were noted from 17 centres of this study comprising 88 isolates exhibiting those increased MICs for CAS (n=59; 5.6%), ANI (n=55; 5.2%) and MCA (n=26; 2.5%). Increased echinocandin MICs towards one, two and three AFAs of this class were displayed by 54/88 (61.4%), 16/88 (18.1%) and 18/88 (20.5%) of the isolates, respectively. Complete echinocandin parallel resistance has been noted following prolonged use of these compounds for treatment of C. albicans and C. parapsilosis infections [33], [34]. Here, simultaneous presence of echinocandinand species-dependent cross-resistance with azoles was found up to 30% depending on candidal species (Table 3 and Table 4). Selection pressure due to continuous exposure appears to play a crucial role in the emergence of azole resistance, thus, high parallel resistance rates for the azoles have to be noted as shown also in this study (8.8% of all isolates). This pattern is aggravated by cross-resistances of azoleresistant isolates to other AFA groups. In previous studies, none of 315 FLC-resistant Candida isolates demonstrated cross-resistance to ANI, whereas cross-resistance to CAS was rarely found (n=4; 1.1%) [35], [36]. In contrast, elevated cross-resistance frequencies of FLC-resistant yeast isolates (n=173) were found in this study for two echinocandins, ANI (n=18; 10.4%) and CAS (n=17; 9.8%). Cross-resistance between azoles and echinocandins, i.e. multi-resistance with different substance classes, may be caused by common resistance mechanisms such as over-expression of genes encoding efflux pumps, multidrug transport systems, lipid-associated membrane (protein) functions and/or membrane fluidity [1], [37], [38], [39]. Complete azole-resistant yeast isolates of this study showed cross-resistance to AMB (n=23; 2.2%) and FCY (n=18; 1.7%). In contrast, cross-resistance of the echinocandin-resistant isolates to AMB (n=4; 0.4%) and FCY (n=1; 0.1%) was much rarer. As determined by SPA, a C. sake isolate showed parallel resistance to all echinocandins and cross-resistance simultaneously to AMB and FCY. Of note, this is the first report to our knowledge of those cross-resistance patterns. Here, testing more than thousand clinical yeast isolates recovered within the course of a multicentre study to eight AFAs, a wide variety of SPs occurring was found. When tested highly standardized in parallel and from the same inoculum, evaluation of different AFA substances by SPA analysis allows detailed insights in the prevalence and distribution of susceptibility patterns of fungal isolates. Since the SPA approach enables a precise description of both known and so far unknown patterns of cross and parallel resistances, it may reflect the resistance situation in a given setting more comprehensively and more detailed compared to data based on standard susceptibility analyses. Consequently, deductions for treatment strategies based on species-specific SPs may be gained for improvement of calculated antifungal chemotherapy.